CALCIUM-DEPENDENT MOLECULES 313
1 : 2 CaM:TFP complex was expected to form, then KAR - 2 was added, results
indicated a competition by TFP and KAR - 2 for the same interdomain binding
site — the second interdomain TFP positive difference peak disappeared and
a negative difference peak at 300 nm appeared indicating KAR - 2 binding.
Using fl uorescence experiments, the reference 83 authors also found that
KAR - 2 and vinblastine can both bind to CaM, forming a ternary complex;
however, both bisindoles bind with reduced affi nity. The combination of CD
and fl uorescence experiments led the authors to conclude that TFP and vin-
blastine compete for the C - terminal CaM binding pocket while KAR - 2 com-
petes with a second TFP molecule for an interdomain CaM site. It can also be
concluded that TFP and vinblastine are calmodulin antagonists competing
with the binding of a target enzyme at the CaM C - terminal hydrophobic
pocket. Additionally, it was found that binding of the target peptide melittin
to calmodulin prevents the binding of both vinblastine and KAR - 2 but not
that of TFP. Binding of target peptides to calmodulin is discussed further in
Section 6.3.2.4 below.
The crystal structure of a 1 : 4 CaM:TFP complex was published in 1994
(PDB: 1LIN).^84 This complex shows three TFP molecules binding in or near
to the C - domain of CaM, along with one TFP molecule binding to the N -
domain. The crystallization mixture contained a 35 : 1 TFP:CaM ratio in con-
trast with the 4.5 : 1 ratio used by the reference 82 authors (PDB: 1CTR),
perhaps explaining the higher TFP occupancy in the crystal. A molecular
dynamics simulation of the CaM:TFP complex in aqueous solution starting
with data from the 1 : 4 CaM:TFP crystal structure was performed by the PDB:
1LIN researchers. The obtained solution structure is very similar to the 1 : 2
CaM:TFP crystal structure discussed above. The computer simulation showed
that the binding ability of the secondary binding site of the C - domain is higher
than that of the primary binding site of the N - domain.
6.3.2.4 Calmodulin–Peptide Binding. In order to carry out its purpose,
calmodulin (CaM) must fi rst bind calcium ions and then attract and bind
peptide segments of its target enzymes in order to activate them (or release
the autoinhibitory effect of the target enzyme CaM - binding peptide). Struc-
tures of Ca 2+ - saturated calmodulin complexed to various peptides have been
studied both in solution by NMR and in the solid state using X - ray crystallog-
raphy. The structures of binding peptides derived from skeletal (skMLCK,
PDB: 2BBM by NMR)^85 and smooth muscle (smMLCK, PDB: 1CDL by X -
ray), 86a as well as from calmodulin kinase II α (CaMKII α , PDB: 1CDM by X -
ray), 86b have been solved. MLCK is the acronym for myosin light chain kinase.
The calmodulin - binding peptides assume random coil structures in solution,
but in the presence of calmodulin they form amphipathic or amphiphilic (con-
taining both polar and nonpolar residues) helices. All of these peptides have
nanomolar (very high) affi nities for calmodulin. Table 6.9 shows the primary
amino acid sequence of some of the calmodulin - binding peptides, and it is
informative to compare them as they are discussed in the following material.